1. Field of the Invention
The present invention relates generally to the fields of cancer and biochemistry. More particularly, it concerns induction of programmed cell death or apoptosis in tumor cells by the p84N5 gene.
2. Description of Related Art
Coordination of cell proliferation and cell death is required for normal development and tissue homeostasis in multicellular organisms. A defect in the normal coordination of these two processes is a fundamental requirement for tumorigenesis. Progression through the cell cycle is highly regulated, requiring the transit of numerous checkpoints (for review, see Hunter, 1993). The extent of cell death is physiologically controlled by activation of a programmed suicide pathway that results in a morphologically recognizable form of death termed apoptosis (Jacobson et al., 1997; Vaux et al., 1994). Both extra-cellular signals, such as tumor necrosis factor, and intracellular signals, like p53, can induce apoptotic cell death. Although many proteins involved in apoptosis or the cell cycle have been identified, the mechanisms by which these two processes are coordinated are not well understood.
Mutation of the retinoblastoma tumor suppressor gene (Rb) alone is sufficient to cause retinoblastoma in humans, suggesting it might play a role in the normal coordination of cell proliferation and cell death (Goodrich and Lee, 1993). The retinoblastoma tumor suppressor protein (p110Rb) can inhibit progression through the G1 phase of the cell cycle (Goodrich et al., 1991). This is accomplished largely by modulation of cellular transcription factors, like E2F1, through direct physical association (Bagchi et al., 1991; Flemington et al., 1993; Kaelin, Jr. et al., 1992; Weintraub et al., 1992). Cyclin-dependent kinase phosphorylation of Rb protein (p110Rb) allows transit through the Rb-enforced checkpoint (Connell-Crowley et al., 1997) by disrupting physical association with these cellular proteins. Several lines of evidence suggest that p110Rb may also regulate apoptosis. Ectopic expression of p110Rb inhibits apoptosis triggered by radiation (Haas-Kogan et al., 1995), E2F1 (Hsieh et al., 1997), p53 (Haupt et al., 1995), myocyte differentiation (Wang et al., 1997), or ceramide (McConkey et al., 1996). Rb protein also is a target for cleavage by caspases during apoptosis (Janicke et al., 1996; An and Dou, 1996). Finally, mouse embryos lacking functional Rb have inappropriately high levels of apoptosis in the central nervous system, the liver, the eye lens, and skeletal muscle (Zacksenhaus et al., 1996). Although these findings suggest that p110Rb may regulate apoptosis, it is unclear whether this regulation is a novel function, or an indirect consequence of Rb-mediated effects on the cell cycle.
The C-terminal half of p110Rb is sufficient for many of its known molecular activities, including modulation of transcription factor function and induction of cell cycle arrest. The purpose of the N-terminal half of p110Rb is undefined. Several observations suggest that this region may be important for normal function. First, some mutations causing low penetrance retinoblastoma specifically alter the N-terminal half of the protein (Dryja et al., 1993; Hogg et al., 1993; Lohmann et al., 1994). Second, the amino acid sequence of the N-terminal half of p110Rb is conserved between mouse, rat, chicken, frog, newt, and human. Finally, N-terminally truncated Rb transgenes are incapable of rescuing developmental defects observed in mice deficient in wild-type Rb (Riley et al., 1997).
The N5 gene was isolated based on its ability to encode a protein that specifically associates with the N-terminal half of p110Rb (Durfee et al., 1994b). Three other proteins, a 70 kDa heat shock protein (Inoue et al., 1995), a kinase (Sterner et al., 1996), and MCM7 (Sterner et al., 1998), have been discovered to bind the N-terminal half of p110Rb. The relevance of these interactions for Rb function is not completely understood, although association of p110Rb with MCM7 does inhibit DNA replication in vitro (Sterner et al., 1998). The N5 protein (p84N5) normally is localized exclusively to the nucleus during interphase and has a region of structural similarity to the death domains of several well-characterized proteins involved in apoptosis, including tumor necrosis factor receptor 1 (TNFR-1) (Feinstein et al., 1995).
Rb gene-associated tumors, including retinoblastomas, small cell lung-carcinoma, osteosarcoma, bladder carcinoma, prostate carcinoma and breast cancer, are a difficult class of tumors to treat. Current therapies are not specific for these tumors and have serious treatment-associated side effects. There exists a need for a treatment that is specific for tumor cells that does not have side effects and specifically targets Rb gene associated tumors.
The present invention concerns methods and compositions for treating cancer in a subject. These methods and compositions utilize the activities associated with the N5 gene product, p84N5. p84N5 contains a functional death domain, can interact with the retinoblastoma gene product, and is normally localized to the nucleus of cells. Increasing the activity level of p84N5 in cancer cells is, therefor, beneficial for the treatment of cancer.
Claimed in the present invention is a viral composition comprising a recombinant viral vector encoding a p84N5 death domain. In other embodiments, the p84N5 death domain may be fused to other protein sequences. Alternatively, the p84N5 may be as shown in SEQ ID NO:1. In preferred embodiments, the recombinant viral vector is an adenoviral, adeno-associated viral, retroviral, herpes viral, papilloma viral, or hepatitus B viral vector.
Also claimed in the present invention is a protein composition comprising purified p84N5 protein. Purified p84N5 contains the p84N5 death domain and the p84N5 protein or portion of said p84N5 protein including a death domain may be fused to a second protein. In preferred embodiments, the p84N5 protein is as shown in SEQ ID NO:2.
Also claimed in the present invention is a recombinant cell exhibiting lower amounts of p84N5 activity when compared to the starting, non-engineered cell, comprising an alteration in transcription, translation, messenger RNA or protein stability, or protein half-life of endogenous p84N5. Alternatively, a recombinant cell exhibiting increased amounts of p84N5 activity when compared to the starting, non-engineered cell, comprising an alteration in transcription, translation, messenger RNA or protein stability, or protein half-life of endogenous p84N5 is claimed. Such cells exhibiting lower or increased amounts of p84N5 activity may be cell lines or cells in transgenic animals.
A method of treating cancer in a subject, comprising contacting said subject with a recombinant vector encoding a p84N5 death domain operably linked to a promoter that functions in said cell. In preferred embodiments, the p84N5 death domain is as shown in SEQ ID NO:1. In preferred embodiments, the recombinant vector encoding a p84N5 death domain is a viral vector. The viral vector may be an adenovirus, adeno-associated virus, retrovirus, herpes virus, papilloma virus, or hepatitus B virus. In preferred embodiments the virus is an adenovirus and is administered to the subject at a dose of about 1010 to about 1012 pfu.
Also claimed in the present invention are methods of treating a subject with cancer with a protein composition comprising purified p84N5. p84N5 may be purified from natural sources or may be recombinant p84N5 produced by a number of means.
Treating cancer is defined as inducing apoptosis, inhibiting cell division, inhibiting metastatic potential, reducing tumor burden, increasing sensitivity to chemotherapy or radiotherapy, killing a cancer cell, inhibiting the growth of a cancer cell, or inducing tumor regression in a subject. In preferred embodiments, the subject is human. The cancer to be treated may be retinoblastoma negative.
In preferred embodiments, treatment of the subject will include a second agent, such as a therapeutic polypeptide, nucleic acid encoding a therapeutic polypeptide, a chemotherapeutic agent, or a radiotherapeutic agent. The second agent may be administered at a different time than the p84N5 treatment. In some embodiments, the second agent may be a nucleic acid encoding p53. Subjects may be treated by intravenous, intraperitoneal, intradermal, intratumoral, intramuscularoral, dermal, nasal, buccal, rectal, vaginal, inhalation, or topical administration of the p84N5 treatment.
The present invention provides the first report that identifies p84N5 as a death domain-containing protein that triggers apoptotic cell death from within the nucleus. Few proteins, other than transcription factors, are known that require nuclear localization to induce apoptosis.
The invention also provides novel mechanisms that transduce nuclear apoptotic signals. The inventors envision that these mechanisms will enable the prediction of tumor responses to genotoxic radiation and/or chemotherapy thereby providing methods for cancer prognosis and new anticancer treatments. Thus, p84N5 induces an ATM-independent, caffeine-sensitive G2/M cell cycle arrest prior to the onset of apoptosis. Furthermore, p84N5-induced apoptosis is preceded by activation of a p53-independent G2/M checkpoint.
The invention also describes identification of the N5 nuclear localization signal and shows that nuclear localization is required to inhibit the clonogenicity of N5 expressing cells. Thus, p84N5 is involved in a Rb-regulated apoptotic pathway that is normally triggered from within the nucleus in response to stimuli, for example, in response to DNA damage. In other studies carried out herein, the inventors have also shown that coexpression of RB can inhibit N5-induced apoptosis, and that their physical association mediates this block.
The inventors have also determined that N5-induced apoptosis is initially accompanied by activation of caspase-6. Activation of caspases-3 and -9 peaks 3 days after the peak of caspase-6 activity. Expression of p84N5 also leads to activation of NF-kB as indicated by nuclear translocation of p65RelA and transcriptional activation of a NF-kB-dependent reporter promoter. Changes are also seen in the relative expression level of Bcl-2 family proteins, including Bak and Bcl-Xs, during p84N5-induced apoptosis. The inventors also demonstrate that p84N5-induced apoptosis does not require p53 and is not inhibited by p53 coexpression.
In another aspect, the inventors demonstrate that expression of p84N5, either by transfection or adenovirus-mediated gene transfer, renders cells inviable. For example, N5 adenovirus infection significantly reduced the proliferation and tumorigenicity of breast, ovarian, and osteosarcoma tumor cell lines. Loss of viability is due to induction of apoptosis. p84N5-expressing cells exhibit many of the typical characteristics of apoptosis, including fragmentation of DNA, exposure of phosphatidylserine on the outer leaflet of the plasma membrane, changes in membrane permeability, and changes in cellular and nuclear morphology. The induction of apoptosis reduced proliferation and tumorigenicity the cancer cells. Thus, N5 encoding nucleic acids provide tools for gene therapy of cancer.
The inventors also demonstrate that co-infection of cancer cells with adenovirus encoding N5 and p53 are highly effective in inducing apoptosis. For example, co-infection of Colo357 X cells with both AdN5 and Adp53 causes a more dramatic reduction growth than infection with either one alone. The invention also explains the mechanism of co-infection as follows; N5 and p53 function in different pathways, N5-induced apoptosis is preceded by a G2/M cell cycle arrest while expression of p53 typically induces a G1 cell cycle arrest. Thus, the invention also provides for anticancer genetherapy using both AdN5 and Adp53.